SUMMARY The majority of head and neck squamous cell carcinoma (HNSCC) patients (~80%) do not respond to immune checkpoint blockade (ICB). Increasing evidence highlights two main barriers to achieving clinical response with immunotherapy in HNSCC patients: (i) the tumor's overall poor antigenicity, which limits the generation of antitumor immunity, and (ii) innate and adaptive immune suppressive mechanisms that result in immune tolerance. The overarching goal of this Program Project (P01) is to address these two barriers to improve the low response rates of HNSCC to immunotherapy. Projects 1 and 2 focus on reprogramming tumor cell antigenicity using two different but complementary approaches and Project 3 focuses on converting these antigenic shifts into ones that provoke successful anti-tumor immunity. This Project (Project 1) focuses on reprogramming the antigenicity of the cancers through epigenetic therapy with the goal of enhancing overall tumor antigen presentation and recognition by the immune system. We hypothesize that a DNA methyltransferase inhibitor can uncover epigenetically silenced genes, such as HLA class I APM components and neoantigens across and within heterogeneous tumor cell populations, to uniformly improve tumor cell antigenicity, and, thus, immunogenicity which translates into effective clinical response with immunotherapy. In our proposal, we leverage a collaborative team science approach with multi-disciplinary expertise in HNSCC, cancer immunology, cancer genomics, epigenetics, bioinformatics, HLA biology, and antigen peptide discovery across multiple institutions and an ongoing phase Ib/II investigator-initiated clinical trial administering a DNA methlytransferase inhibitor in combination with ICB in HNSCC patients. Utilizing state of the art technology such as DNA methylation, bulk RNASeq and single-cell RNASeq (scRNASeq) platforms, we plan to comprehensively characterize the pre- and on-treatment biopsy samples to assess changes in expression levels of the HLA class I APM components and identify a panel of shared neoantigen(s) expressed within and across HNSCCs in order to determine whether we are able to successful reprogram the antigenicity of a tumor through epigenetic therapy.